(1) Field of the Invention
The present invention relates to the use of polypeptides which prevent freezing damage to materials and which are derived from cDNA's of RNA's isolated from Arabidopsis thaliana which have been exposed to near freezing temperatures or which have been treated with abscisic acid. The resulting polypeptides are cryoprotective to chemical and biological materials. In particular the present invention relates to a preferred 15 kilodalton polypeptide, referred to as COR15, which is cryoprotective.
(2) Prior Art
In many species of higher plants, a period of exposure to low non-freezing temperatures results in an increased level of freezing tolerance (Thomashow, M. F., Adv. Genet. 28: 99-131 (1990)). Davies FASEB Journal 4, 2460-2468 (1990) describes fish antifreeze proteins. Considerable effort has been directed at understanding the molecular basis of this cold acclimation response, yet the mechanism remains poorly understood. A large number of biochemical changes have been shown to be associated with cold acclimation including alterations in lipid composition, increased sugar and soluble protein content, and the appearance of new isozymes (Thomashow, M. F., Adv. Genet. 28: 99-131 (1990); and Steponkus, P. L., Cold acclimation and freezing injury from a perspective of the plasma membrane. In Katterman, F. (ed), Environmental Injury to Plants pp 1-16. Academic Press, San Diego (1990)). However, in most cases, the exact role and relative importance that a given change has in cold acclimation remains uncertain. A notable exception regards certain changes in lipid composition. Steponkus and colleagues (Steponkus, P. L., et al., Proc. Natl. Acad. Sci. USA 85: 9026-9030 (1988)) have presented data indicating that specific changes in the phospholipid composition of rye cell plasma membranes dramatically alters the cryobehavior of the membranes and contributes directly to the increased freezing tolerance of acclimated cells.
In 1970, Weiser (Weiser, C. J., Science 169: 1269-1278 (1970)) suggested that cold acclimation might involve changes in gene expression. Since then, it has been clearly established that changes in gene expression occur during cold acclimation in a wide range of plant species (Thomashow, M. F., Adv. Genet. 28: 99-131 (1990) and Thomashow, M. F., et al, Horticultural Biotech 305-314 (1990)). Efforts are now being directed at determining the functions of these cold-regulated genes. One interesting possibility is suggested by the work of Heber and colleagues (Volger, H. G., et al., Biochim. Biophys. Acta 412: 335-349 (1975); Hincha, D. K., et al., Plant Physiol. Biochem. 27: 795-801 (1989); Hincha, D. K., et al., Planta 180: 416-419 (1990)). These investigators have shown that cold acclimated spinach and cabbage, but not nonacclimated plants, synthesize hydrophilic, heat-stable, low molecular weight polypeptides (10-20 kd) that have cryoprotective properties. In particular, these polypeptides, which have not been purified or individually identified, were reported to be greater than 10,000 times (molar basis) more effective in protecting thylakoid membranes against freezing damage in an in vitro assay than were known low molecular weight cryoprotectants such as sucrose. Whether these polypeptides contribute significantly to the increased freezing tolerance of cold acclimated plants in vivo remains to be determined.
It has been established that Arabidopsis becomes more tolerant to freezing temperatures when exposed to low non-freezing temperatures and that changes in gene expression occur during the cold acclimation process (Gilmour, S. J., et al., Plant Physiol. 97: 745-750 (1988)). It has been reported that four Arabidopsis cor (cold-regulated) genes encode polypeptides, 160, 47, 24, and 15 kDa in mass, that share an unusual biochemical property: they remain soluble upon boiling in aqueous solution (Lin, C., et al., Plant Physiol. 94: 1078-1083 (1990)). In addition, it has been found that wheat also has cot genes that encode "boiling-stable" polypeptides and that wheat has a cor gene(s) that is related to Arabidopsis COR47, a cold-regulated gene that encodes a 47 kDa boiling-stable polypeptide (Lin, C., et al., Plant Physiol. 94: 1078-1083 (1990)). It has been suggested it is likely that the boiling-stable COR polypeptides of Arabidopsis and wheat have a fundamental role in plants acclimating to cold temperatures (Lin, C., et al., Plant Physiol. 94: 1078-1083 (1990)). In addition, it has been speculated (Lin, C., et al., Plant Physiol. 94: 1078-1083 (1990)) that these polypeptides might be analogous to the cryoprotective polypeptides described by Heber and colleagues (Volger, H. G., et al., Biochim. Biophys. Acta 412:335-349 (1975); Hincha, D. K., et al., Plant Physiol. Biochem. 27:795-801 (1989); and Hincha, D. K., et al., Planta 180: 416-419 (1990)).
There has been no suggestion that the genes encoding these cold "regulated" polypeptides might be synthesized and used to protect various materials. The problem has been to provide DNA which encodes the polypeptides.